Lettuce named hmx 7555

ABSTRACT

A novel romaine lettuce cultivar, designated HMX 7555, is disclosed. The invention relates to the seeds of lettuce cultivar HMX 7555, to the plants of lettuce line HMX 7555 and to methods for producing a lettuce plant by crossing the cultivar HMX 7555 with itself or another lettuce line. The invention further relates to methods for producing a lettuce plant containing in its genetic material one or more transgenes and to the transgenic plants produced by that method and to methods for producing other lettuce lines derived from the cultivar HMX 7555.

BACKGROUND OF THE INVENTION

[0001] The present invention relates to a new and distinctive Romainelettuce (Lactuca sativa) variety, designated HMX 7555. There arenumerous steps in the development of any novel, desirable plantgermplasm. Plant breeding begins with the analysis and definition ofproblems and weaknesses of the current germplasm, the establishment ofprogram goals, and the definition of specific breeding objectives. Thenext step is selection of germplasm that possess the traits to meet theprogram goals. The goal is to combine in a single variety or hybrid animproved combination of desirable traits from the parental germplasm.These important traits may include increased head size and weight,higher seed yield, improved color, resistance to diseases and insects,tolerance to drought and heat, and better agronomic quality.

[0002] Practically speaking, all cultivated forms of lettuce belong tothe highly polymorphic species Lactuca sativa that is grown for itsedible head and leaves. As a crop, lettuces are grown commerciallywherever environmental conditions permit the production of aneconomically viable yield. Lettuce is the world's most popular salad. Inthe United States, the principal growing regions are California andArizona which produce approximately 287,000 acres out of a total annualacreage of more than 300,000 acres (USDA, 2001). Fresh lettuces areavailable in the United States year-round although the greatest supplyis from May through October. For planting purposes, the lettuce seasonis typically divided into three categories, early, mid and late, withthe coastal areas planting from January to August, and the desertregions from August to December. Fresh lettuces are consumed nearlyexclusively as fresh, raw product, occasionally as a cooked vegetable. .

[0003]Lactuca sativa is in the Cichoreae tribe of the Asteraceae(Compositae family). Lettuce is related to chicory, sunflower, aster,dandelion, artichoke and chrysanthemum. Sativa is one of about 300species in the genus Lactuca. There are seven different morphologicaltypes of lettuces: the Crisphead group includes the iceberg and bataviantypes. Iceberg lettuce has a large, firm head with a crisp texture and awhite or creamy yellow interior. The Batavian lettuce predates theiceberg type and has a smaller and less firm head. The Butterhead grouphas a small, soft head with an almost oily texture. The Romaine, alsoknown as cos lettuce, has elongated upright leaves forming a loose, loafshaped head. The outer leaves are usually dark green. The Leaf lettucescomes in many varieties, none of which form a head. The next three typesare seldom seen in the United States: Latin lettuce looks like a crossbetween romaine and butterhead; stem lettuce has long, narrow leaves andthick, edible stems. Oilseed lettuce, finally, is a primitive type grownfor its large seeds that are pressed to obtain oil.

[0004]Lactuca sativa is a simple diploid species with nine pairs ofchromosomes (2n=2x=18). Lettuce is an obligate self-pollinating species.This means that the pollen is shed before stigma emergence, assuring100% self-fertilization. Since each lettuce flower is an aggregate ofabout 10-20 individual florets (typical of the Compositae family),manual removal of the anther tubes containing the pollen is tedious. Assuch, a - modified method of misting to wash the pollen off prior tofertilization is needed to assure crossing or hybridization. About 60-90min past sunrise, flowers to be used for crossings are selected. Thebasis for selection are open flowers, with the stigma emerged and thepollen visibly attached to the single stigma (about 10-20 stigma). Using3-4 pumps of water from a regular spray bottle, the pollen grains arewashed off with enough pressure to dislodge the pollen grains, but notenough to damage the style. Excess water is dried off with clean papertowels. About 30 min later the styles should spring back up and the twolobes of the stigma are visibly open in a “V” shape. Pollens fromanother variety or donor parent are then introduced by gently rubbingthe stigma and style of the donor parent to the maternal parent. Tagswith the pertinent information on date and pedigree are then secured tothe flowers

[0005] Choice of breeding or selection methods depends on the mode ofplant reproduction, the heritability of the trait(s) being improved, andthe type of cultivar used commercially (e.g., F₁ hybrid cultivar,pureline cultivar, etc.). For highly heritable traits, a choice ofsuperior individual plants evaluated at a single location will beeffective, whereas for traits with low heritability, selection should bebased on mean values obtained from replicated evaluations of families ofrelated plants. Popular selection methods commonly include pedigreeselection, modified pedigree selection, mass selection, and recurrentselection.

[0006] The complexity of inheritance influences choice of the breedingmethod. Backcross breeding is used to transfer one or a few favorablegenes for a highly heritable trait into a desirable cultivar. Thisapproach has been used extensively for breeding disease-resistantcultivars. Various recurrent selection techniques are used to improvequantitatively inherited traits controlled by numerous genes. The use ofrecurrent selection in self-pollinating crops depends on the ease ofpollination, the frequency of successful hybrids from each pollination,and the number of hybrid offspring from each successful cross

[0007] Each breeding program should include a periodic, objectiveevaluation of the efficiency of the breeding procedure. Evaluationcriteria vary depending on the goal and objectives, but should includegain from selection per year based on comparisons to an appropriatestandard, overall value of the advanced breeding lines, and number ofsuccessful cultivars produced per unit of input (e.g., per year, perdollar expended, etc.).

[0008] Promising advanced breeding lines are thoroughly tested andcompared to appropriate standards in environments representative of thecommercial target area(s) for three years at least. The best lines arecandidates for new commercial cultivars; those still deficient in a fewtraits are used as parents to produce new populations for furtherselection.

[0009] These processes, which lead to the final step of marketing anddistribution, usually take from eight to 12 years from the time thefirst cross is made. Therefore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

[0010] A most difficult task is the identification of individuals thatare genetically superior, because for most traits the true genotypicvalue is masked by other confounding plant traits or environmentalfactors. One method of identifying a superior plant is to observe itsperformance relative to other experimental plants and to a widely grownstandard cultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

[0011] The goal of plant breeding is to develop new, unique and superiorlettuce cultivars. The breeder initially selects and crosses two or moreparental lines, followed by repeated selfing and selection, producingmany new genetic combinations. The breeder can theoretically generatebillions of different genetic combinations via crossing, selfing andmutations. The breeder has no direct control at the cellular level.Therefore, two breeders will never develop the same line, or even verysimilar lines, having the same lettuce traits.

[0012] Each year, the plant breeder selects the germplasm to advance tothe next generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The cultivarsthat are developed are unpredictable. This unpredictability is becausethe breeder's selection occurs in unique environments, with no controlat the DNA level (using conventional breeding procedures), and withmillions of different possible genetic combinations being generated. Abreeder of ordinary skill in the art cannot predict the final resultinglines he develops, except possibly in a very gross and general fashion.The same breeder cannot produce the same line twice by using the exactsame original parents and the same selection techniques. Thisunpredictability results in the expenditure of large research monies todevelop superior lettuce cultivars.

[0013] The development of commercial lettuce cultivars requires thedevelopment of lettuce varieties, the crossing of these varieties, andthe evaluation of the crosses. Pedigree breeding and recurrent selectionbreeding methods are used to develop cultivars from breedingpopulations. Breeding programs combine desirable traits from two or morevarieties or various broad-based sources into breeding pools from whichcultivars are developed by selfing and selection of desired phenotypes.The new cultivars are crossed with other varieties and the hybrids fromthese crosses are evaluated to determine which have commercialpotential.

[0014] Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents which possess favorable, complementary traits, are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁'s or by intercrossing two F₁'s (sib mating). Selection of the bestindividuals is usually begun in the F₂ population; then, beginning inthe F₃, the best individuals in the best families are selected.Replicated testing of families, or hybrid combinations involvingindividuals of these families, often follows in the F₄ generation toimprove the effectiveness of selection for traits with low heritability.At an advanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars.

[0015] Mass and recurrent selections can be used to improve populationsof either self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

[0016] Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror line that is the recurrent parent. The source of the trait to betransferred is called the donor parent. The resulting plant is expectedto have the attributes of the recurrent parent (e.g., cultivar) and thedesirable trait transferred from the donor parent. After the initialcross, individuals possessing the phenotype of the donor parent areselected and repeatedly crossed (backcrossed) to the recurrent parent.The resulting plant is expected to have the attributes of the recurrentparent (e.g., cultivar) and the desirable trait transferred from thedonor parent.

[0017] The single-seed descent procedure in the strict sense refers toplanting a segregating population, harvesting a sample of one seed perplant, and using the one-seed sample to plant the next generation. Whenthe population has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population, will be represented by a progenywhen generation advance is completed

[0018] Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., “Principles of Plant Breeding” John Wiley and Son,pp.115-161,1960; Allard, 1960; Simmonds, 1979; Sneep et al., 1979; Fehr,1987).

[0019] Proper testing should detect any major faults and establish thelevel of superiority or improvement over current cultivars. In additionto showing superior performance, there must be a demand for a newcultivar that is compatible with industry standards or which creates anew market. The introduction of a new cultivar will incur additionalcosts to the seed producer, the grower, processor and consumer; forspecial advertising and marketing, altered seed and commercialproduction practices, and new product utilization. The testing precedingrelease of a new cultivar should take into consideration research anddevelopment costs as well as technical superiority of the finalcultivar. For seed-propagated cultivars, it must be feasible to produceseed easily and economically.

[0020] Lettuce in general and romaine lettuce in particular is animportant and valuable vegetable crop. Thus, a continuing goal of plantbreeders is to develop stable, high yielding lettuce cultivars that areagronomically sound. The reasons for this goal are obviously to maximizethe amount of yield produced on the land. To accomplish this goal, thelettuce breeder must select and develop lettuce plants that have thetraits that result in superior cultivars.

SUMMARY OF THE INVENTION

[0021] According to the invention, there is provided a novel romainelettuce cultivar, designated HMX 7555. This invention thus relates tothe seeds of lettuce cultivar HMX 7555, to the plants of lettucecultivar HMX 7555 and to methods for producing a lettuce plant producedby crossing the lettuce HMX 7555 with itself or another lettuce line,and to methods for producing a lettuce plant containing in its geneticmaterial one or more transgenes and to the transgenic lettuce plantsproduced by that method. This invention also relates to methods forproducing other lettuce cultivars derived from lettuce cultivar HMX 7555and to the lettuce cultivar derived by the use of those methods. Thisinvention further relates to hybrid lettuce seeds and plants produced bycrossing the line HMX 7555 with another lettuce line.

[0022] In another aspect, the present invention provides regenerablecells for use in tissue culture of lettuce cultivar HMX 7555. The tissueculture will preferably be capable of regenerating plants having thephysiological and morphological characteristics of the foregoing lettuceplant, and of regenerating plants having substantially the same genotypeas the foregoing lettuce plant. Preferably, the regenerable cells insuch tissue cultures will be embryos, protoplasts, seeds, callus,pollen, leaves, anthers, roots, and meristematic cells. Still further,the present invention provides lettuce plants regenerated from thetissue cultures of the invention.

[0023] Another objective of the invention is to provide methods forproducing other lettuce plants derived from lettuce cultivar HMX 7555.Lettuce cultivars derived by the use of those methods are also part ofthe invention.

[0024] The invention also relates to methods for producing a lettuceplant containing in its genetic material one or more transgenes and tothe transgenic lettuce plant produced by that method.

[0025] In another aspect, the present invention provides for single geneconverted plants of HMX 7555. The single transferred gene may preferablybe a dominant or recessive allele. Preferably, the single transferredgene will confer such trait as male sterility, herbicide resistance,insect resistance, resistance for bacterial, fungal, or viral disease,male fertility, enhanced nutritional quality and industrial usage. Thesingle gene may be a naturally occurring lettuce gene or a transgeneintroduced through genetic engineering techniques.

[0026] The invention further provides methods for developing lettuceplant in a lettuce plant breeding program using plant breeding techniqueincluding recurrent selection, backcrossing, pedigree breeding,restriction fragment length polymorphism enhanced selection, geneticmarker enhanced selection and transformation. Seeds, lettuce plant, andparties thereof produced by such breeding methods are also part of theinvention.

DEFINITIONS

[0027] In the description and tables which follow, a number of terms areused. In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

[0028] Allele. The allele is any of one or more alternative form of agene, all of which alleles relates to one trait or characteristic. In adiploid cell or organism, the two alleles of a given gene occupycorresponding loci on a pair of homologous chromosomes.

[0029] Backcrossing. Backcrossing is a process in which a breederrepeatedly crosses hybrid progeny back to one of the parents, forexample, a first generation hybrid F₁ with one of the parental genotypeof the F₁ hybrid.

[0030] Essentially all the physiological and morphologicalcharacteristics. A plant having essentially all the physiological andmorphological characteristics means a plant having the physiological andmorphological characteristics, except for the characteristics derivedfrom the converted gene.

[0031] Regeneration. Regeneration refers to the development of a plantfrom tissue culture.

[0032] Single gene converted. Single gene converted or conversion plantrefers to plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of a line are recovered in addition tothe single gene transferred into the line via the backcrossing techniqueor via genetic engineering.

[0033] Maturity Date. Maturity refers to the stage when the plants areof full size or optimum weight, in marketable form or shape to be ofcommercial or economic value. In romaine types they range from 50-75days from time of seeding, depending upon the season of the year.

[0034] RHS. RHS refers to the Royal Horticultural Society of Englandwhich publishes an official botanical color chart quantitativelyidentifying colors according to a defined numbering system, The chartmay be purchased from Royal Hort Society Enterprise Ltd RHS Garden;Wisley, Woking; Surrey GU236QB, UK.

[0035] Lettuce Yield (Tons/Acre). The yield in tons/acre is the actualyield of the lettuce at harvest.

DETAILED DESCRIPTION OF THE INVENTION

[0036] Lettuce cultivar HMX 7555 has superior characteristics and wasdeveloped from the cross 95P054(female) and 95P060 (male), which wasmade in the fall of 1995 in the greenhouse at Harris Moran Research inCalifornia. The F₁ hybrids (95V205) were grown in a greenhouse duringthe winter of 1996. F₂ selection, plot number 96D277, was made at SanJuan Bautista, Calif. in the spring of 1996. The F₃ selections were madein the spring of 1997 at San Juan Bautista, Calif. F₄ plants wereselected in a field plot in California in September 1998; F₅ selectionswere made in the spring of 1999 in Yuma, Ariz.; F₆ generation was bulkedin field plots at Davis, Calif.,. F₇ plants were selected in a fieldplot at San Juan Bautista, Calif. in Summer. F₈ plants were selected andbulked in field plots near Los Mochis, Sinaloa, Mexico in February,1999. The F₉ generation is a stock seed increase at San Juan Bautista,Calif.

[0037] The cultivar HMX 7555 is most similar to ‘Green Towers’ and‘Darkland Cos’ cultivars but differs in that HMX 7555 is darker thanGreen Towers (RHS 137A vs. RHS 137C, respectively). HMX 7555 has also aheavier frame (1837 g) than Darkland Cos (1523 g) as well as fasterbolting under hot conditions (97 days vs. 112 days, respectively). Underwarm conditions, HMX 7555 averaged heavier heads than Green Towers (1121g vs, 1023 g respectively). In cooler conditions, HMX 7555 performs alsobetter at 1837 g vs. 1689 g for Green Towers.

[0038] HMX 7555 is a romaine lettuce with attractive, dark, glossy,leaves. It has a large, fully bodied head, with superior weight. HMX7555 is resistant to downy mildew pathotype I and exhibits anintermediate resistance to Sclerotinia rot. HMX 7555 has shown a verygood adaptability in Southwest, West Coast and Northeast regions of theUnited states.

[0039] Some of the criteria used to select in various generationsinclude: color, disease resistances, head weight, number of leaves,appearance and length, yield, emergence, maturity, plant architecture,seed yield and quality, and disease resistance

[0040] The cultivar has shown uniformity and stability for the traits,within the limits of environmental influence for the traits. It has beenself-pollinated a sufficient number of generations with carefulattention to uniformity of plant type. The line has been increased withcontinued observation for uniformity. No variant traits have beenobserved or are expected in HMX 7555.

[0041] Lettuce cultivar HMX 7555 has the following morphologic and othercharacteristics (based primarily on data collected at San Juan Bautista,Ca, Research Station, and field plots at Yuma, Ariz.).

VARIETY DESCRIPTION INFORMATION

[0042] Plant Type

[0043] Romaine.

[0044] Seed

[0045] Color: white

[0046] Light dormancy: light not required

[0047] Heat dormancy: susceptible

[0048] Cotyledon to Fourth Leaf Stage

[0049] Shape of cotyledons: spatulate

[0050] Undulation: flat

[0051] Anthocyanin distribution: absent

[0052] Rolling: absent

[0053] Cupping: uncupped

[0054] Reflexing: none

[0055] Mature Leaves

[0056] Margin—Incision depth: absent/shallow

[0057] Margin—Indentation:entire

[0058] Margin—Undulation of the apical margin: absent/slight

[0059] Green color: very dark green

[0060] Anthocyanin—Distribution: absent

[0061] Size: large

[0062] Glossiness: dull

[0063] Blistering: absent/slight

[0064] Trichomes: absent

[0065] Leaf thickness: thick

[0066] Plant (at Market Stage)

[0067] Head shape: non heading

[0068] Head size class: large

[0069] Head weight: 1221 g

[0070] Head firmness: loose

[0071] Core

[0072] Diameter at base of head: 34 mm

[0073] Core height from base of head to apex: 81 mm

[0074] Maturity

[0075] Summer: 103 days, 5 days earlier than Green Towers

[0076] Winter: 91 days, 5 days earlier than Green Towers

[0077] Adaptation

[0078] Primary Regions of Adaptation (tested and proven adapted)

[0079] Southwest (California, Arizona desert): adapted

[0080] West Coast: adapted

[0081] Southeast: Not tested

[0082] Northeast: adapted

[0083] Spring area—Salinas Valley, Santa Maria, Calif.

[0084] Summer area—Salinas Valley, Santa Maria, Calif.

[0085] Fall area—Yuma, Ariz.

[0086] Winter area—Yuma, Ariz.

[0087] Greenhouse: Adapted

[0088] Soil Type: Mineral

[0089] Disease and Stress Reactions

[0090] Virus

[0091] Big Vein: Susceptible

[0092] Lettuce Mosaic: Susceptible

[0093] Cucumber Mosaic: Not tested

[0094] Broad Bean Wilt: Not tested

[0095] Turnip Mosaic: Not tested

[0096] Best Western Yellows: Not tested

[0097] Lettuce Infectious Yellows: Not tested

[0098] Fungal/Bacterial

[0099] Corky Root Rot (Pythium Root Rot): susceptible

[0100] Downy Mildew: Resistant to pathotype 1

[0101] Powdery Mildew: Susceptible

[0102] Sclerotinia Rot: Intermediate

[0103] Bacterial Soft Rot (Pseudomonas spp. & others): Not tested

[0104] Botrytis (Gray Mold): Not tested.

[0105] Insects

[0106] Cabbage Loopers: Not tested

[0107] Root Aphids: Not tested

[0108] Green Peach Aphid: Not tested

[0109] Physiological/Stress

[0110] Tipburn: Intermediate

[0111] Heat: Intermediate

[0112] Drought: Not tested

[0113] Cold: Intermediate

[0114] Salt: Not tested

[0115] Brown Rib: Not tested

[0116] Post Harvest

[0117] Pink Rib: Not tested

[0118] Russet Spotting: Not tested

[0119] Rusty Brown Discoloration: Not tested

[0120] Internal Rib Necrosis (Blackheart, Gray Rib, Gray Streak): Nottested

[0121] Brown Stain: Not tested

FURTHER EMBODIMENTS OF THE INVENTION

[0122] This invention also is directed to methods for producing alettuce cultivar plant by crossing a first parent lettuce plant with asecond parent lettuce plant wherein either the first or second parentlettuce plant is an lettuce plant of the line HMX 7555. Further, bothfirst and second parent lettuce plants can come from the cultivar HMX7555. Still further, this invention also is directed to methods forproducing a cultivar HMX 7555-derived lettuce plant by crossing cultivarHMX 7555 with a second lettuce plant and growing the progeny seed, andrepeating the crossing and growing steps with the cultivar HMX7555-derived plant from 0 to 7 times. Thus, any such methods using thecultivar HMX 7555 are part of this invention: selfing, backcrosses,hybrid production, crosses to populations, and the like. All plantsproduced using cultivar HMX 7555 as a parent are within the scope ofthis invention, including plants derived from cultivar HMX 7555.Advantageously, the cultivar is used in crosses with other, different,cultivars to produce first generation (F₁) lettuce seeds and plants withsuperior characteristics.

[0123] As used herein, the term plant includes plant cells, plantprotoplasts, plant cell tissue cultures from which lettuce plants can beregenerated, plant calli, plant clumps and plant cells that are intactin plants or parts of plants, such as embryos, pollen, ovules, flowers,seeds, roots, anthers, and the like.

[0124] As is well known in the art, tissue culture of lettuce can beused for the in vitro regeneration of a lettuce plant. Tissue culture ofvarious tissues of lettuces and regeneration of plants therefrom is wellknown and widely published. For example, reference may be had to Teng etal., HortScience. 1992, 27: 9,1030-1032 Teng et al., HortScience. 1993,28: 6, 669-1671, Zhang et al., Journal of Genetics and Breeding. 1992,46: 3, 287-290, Webb et al., Plant Cell Tissue and Organ Culture. 1994,38: 1, 77-79, Curtis et al., Journal of Experimental Botany. 1994, 45:279,1441-1449, Nagata et al., Journal for the American Society forHorticultural Science. 2000,125: 6, 669-672. It is clear from theliterature that the state of the art is such that these methods ofobtaining plants are, and were, “conventional” in the sense that theyare routinely used and have a very high rate of success. Thus, anotheraspect of this invention is to provide cells which upon growth anddifferentiation produce lettuce plants having the physiological andmorphological characteristics of variety HMX 7555.

[0125] With the advent of molecular biological techniques that haveallowed the isolation and characterization of genes that encode specificprotein products, scientists in the field of plant biology developed astrong interest in engineering the genome of plants to contain andexpress foreign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes”. Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed line.

[0126] Plant transformation involves the construction of an expressionvector that will function in plant cells. Such a vector comprises DNAcomprising a gene under control of or operatively linked to a regulatoryelement (for example, a promoter). The expression vector may contain oneor more such operably linked gene/regulatory element combinations. Thevector(s) may be in the form of a plasmid, and can be used alone or incombination with other plasmids, to provide transformed lettuce plants,using transformation methods as described below to incorporatetransgenes into the genetic material of the lettuce plant(s).

[0127] Expression Vectors for Lettuce Transformation

[0128] Marker Genes—Expression vectors include at least one geneticmarker, operably linked to a regulatory element (a promoter, forexample) that allows transformed cells containing the marker to beeither recovered by negative selection, i.e., inhibiting growth of cellsthat do not contain the selectable marker gene, or by positiveselection, i.e., screening for the product encoded by the geneticmarker. Many commonly used selectable marker genes for planttransformation are well known in the transformation arts, and include,for example, genes that code for enzymes that metabolically detoxify aselective chemical agent which may be an antibiotic or a herbicide, orgenes that encode an altered target which is insensitive to theinhibitor. A few positive selection methods are also known in the art.

[0129] One commonly used selectable marker gene for plant transformationis the neomycin phosphotransferase II (nptII) gene, isolated fromtransposon Tn5, which when placed under the control of plant regulatorysignals confers resistance to kanamycin. Fraley et al., Proc. Natl.Acad. Sci. U.S.A., 80:4803 (1983). Another commonly used selectablemarker gene is the hygromycin phosphotransferase gene which confersresistance to the antibiotic hygromycin. Vanden Elzen et al., Plant Mol.Biol., 5:299 (1985).

[0130] Additional selectable marker genes of bacterial origin thatconfer resistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant. Hayford et al., Plant Physiol.86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987), Svab etal., Plant Mol. Biol. 14:197 (1990< Hille et al., Plant Mol. Biol. 7:171(1986). Other selectable marker genes confer resistance to herbicidessuch as glyphosate, glufosinate or broxynil. Comai et al., Nature317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618 (1990) andStalker et al., Science 242:419-423 (1988).

[0131] Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13:67(1987), Shah et al., Science 233:478 (1986), Charest et al., Plant CellRep. 8:643 (1990).

[0132] Another class of marker genes for plant transformation requirescreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include â-glucuronidase (GUS,â-galactosidase, luciferase and chloramphenicol, acetyltransferase.Jefferson, R. A., Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al., EMBOJ. 8:343 (1989), Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131(1987), DeBlock et al., EMBO J. 3:1681 (1984).

[0133] Recently, in vivo methods for visualizing GUS activity that donot require destruction of plant tissue have been made available.Molecular Probes publication 2908, Imagene Green™, p. 1-4 (1993) andNaleway et al., J. Cell Biol. 115:151 a (1991). However, these in vivomethods for visualizing GUS activity have not proven useful for recoveryof transformed cells because of low sensitivity, high fluorescentbackgrounds and limitations associated with the use of luciferase genesas selectable markers.

[0134] More recently, a gene encoding Green Fluorescent Protein (GFP)has been utilized as a marker for gene expression in prokaryotic andeukaryotic cells. Chalfie et al., Science 263:802 (1994). GFP andmutants of GFP may be used as screenable markers.

[0135] Promoters—Genes included in expression vectors must be driven bynucleotide sequence comprising a regulatory element, for example, apromoter. Several types of promoters are now well known in thetransformation arts, as are other regulatory elements that can be usedalone or in combination with promoters.

[0136] As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred”.Promoters which initiate transcription only in certain tissue arereferred to as “tissue-specific”. A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

[0137] A. Inducible Promoters

[0138] An inducible promoter is operably linked to a gene for expressionin lettuce. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in lettuce. With an inducible promoter the rateof transcription increases in response to an inducing agent.

[0139] Any inducible promoter can be used in the instant invention. SeeWard et al., Plant Mol. Biol. 22:361-366 (1993). Exemplary induciblepromoters include, but are not limited to, that from the ACEI systemwhich responds to copper (Meft et al., PNAS 90:4567-4571 (1993)); In2gene from maize which responds to benzenesulfonamide herbicide safeners(Hershey et al., Mol. Gen Genetics 227:229-237 (1991) and Gatz et al.,Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz etal., Mol. Gen. Genetics 227:229-237 (1991). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena etal., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991).

[0140] B. Constitutive Promoters

[0141] A constitutive promoter is operably linked to a gene forexpression in lettuce or the constitutive promoter is operably linked toa nucleotide sequence encoding a signal sequence which is operablylinked to a gene for expression in lettuce.

[0142] Many different constitutive promoters can be utilized in theinstant invention. Exemplary constitutive promoters include, but are notlimited to, the promoters from plant viruses such as the 35S promoterfrom CaMV (Odell et al., Nature 313:810-812 (1985) and the promotersfrom such genes as rice actin (McElroy et al., Plant Cell 2:163-171(1990)); ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632(1989) and Christensen et al., Plant Mol. Biol. 18:675-689 (1992)); PEMU(Last et al., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten etal., EMBO J. 3:2723-2730 (1984)) and maize H3 histone (Lepetit et al.,Mol. Gen. Genetics 231:276-285 (1992) and Atanassova et al., PlantJournal 2 (3): 291-300 (1992)).

[0143] The ALS promoter, Xba1/NcoI fragment 5′ to the Brassica napusALS3 structural gene (or a nucleotide sequence similarity to saidXba1/NcoI fragment), represents a particularly useful constitutivepromoter. See PCT application WO96/30530.

[0144] C. Tissue-Specific or Tissue-Preferred Promoters

[0145] A tissue-specific promoter is operably linked to a gene forexpression in lettuce. Optionally, the tissue-specific promoter isoperably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a gene for expression in lettuce. Plantstransformed with a gene of interest operably linked to a tissue-specificpromoter produce the protein product of the transgene exclusively, orpreferentially, in a specific tissue.

[0146] Any tissue-specific or tissue-preferred promoter can be utilizedin the instant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai et al., Science 23:476-482(1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A.82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985)and Timko et al., Nature 318:579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics217:240-245 (1989)); a pollen-specific promoter such as that from Zm13(Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993)) or amicrospore-preferred promoter such as that from apg (Twell et al., Sex.Plant Reprod. 6:217-224 (1993).

[0147] Signal Sequences for Targeting Proteins to SubcellularCompartments

[0148] Transport of protein produced by transgenes to a subcellularcompartment such as the chloroplast, vacuole, peroxisome, glyoxysome,cell wall or mitochondroin or for secretion into the apoplast, isaccomplished by means of operably linking the nucleotide sequenceencoding a signal sequence to the 5′ and/or 3′ region of a gene encodingthe protein of interest. Targeting sequences at the 5′ and/or 3′ end ofthe structural gene may determine, during protein synthesis andprocessing, where the encoded protein is ultimately compartmentalized.

[0149] The presence of a signal sequence directs a polypeptide to eitheran intracellular organelle or subcellular compartment or for secretionto the apoplast. Many signal sequences are known in the art. See, forexample Becker et al., Plant Mol. Biol. 20:49 (1992), Close, P. S.,Master's Thesis, Iowa State University (1993), Knox, C., et al.,“Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley”, Plant Mol. Biol. 9:3-17 (1987), Lerner et al., Plant Physiol.91:124-129 (1989), Fontes et al., Plant Cell 3:483-496 (1991), Matsuokaet al., Proc. Natl. Acad. Sci. 88:834 (1991), Gould et al., J. Cell.Biol. 108:1657 (1989), Creissen et al., Plant J. 2:129 (1991), Kalderon,et al., A short amino acid sequence able to specify nuclear location,Cell 39:499-509 (1984), Steifel, et al., Expression of a maize cell wallhydroxyproline-rich glycoprotein gene in early leaf and root vasculardifferentiation, Plant Cell 2:785-793 (1990).

[0150] Foreign Protein Genes and Agronomic Genes

[0151] With transgenic plants according to the present invention, aforeign protein can be produced in commercial quantities. Thus,techniques for the selection and propagation of transformed plants,which are well understood in the art, yield a plurality of transgenicplants which are harvested in a conventional manner, and a foreignprotein then can be extracted from a tissue of interest or from totalbiomass. Protein extraction from plant biomass can be accomplished byknown methods which are discussed, for example, by Heney and Orr, Anal.Biochem. 114:92-6 (1981).

[0152] According to a preferred embodiment, the transgenic plantprovided for commercial production of foreign protein is lettuce. Inanother preferred embodiment, the biomass of interest is seed. For therelatively small number of transgenic plants that show higher levels ofexpression, a genetic map can be generated, primarily via conventionalRFLP, PCR and SSR analysis, which identifies the approximate chromosomallocation of the integrated DNA molecule. For exemplary methodologies inthis regard, see Glick and Thompson, Methods in Plant Molecular Biologyand Biotechnology CRC Press, Boca Raton 269:284 (1993). Map informationconcerning chromosomal location is useful for proprietary protection ofa subject transgenic plant. If unauthorized propagation is undertakenand crosses made with other germplasm, the map of the integration regioncan be compared to similar maps for suspect plants, to determine if thelatter have a common parentage with the subject plant. Map comparisonswould involve hybridizations, RFLP, PCR, SSR and sequencing, all ofwhich are conventional techniques.

[0153] Likewise, by means of the present invention, agronomic genes canbe expressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

[0154] 1. Genes that Confer Resistance to Pests or Disease and thatEncode:

[0155] A. Plant disease resistance genes. Plant defenses are oftenactivated by specific interaction between the product of a diseaseresistance gene (R) in the plant and the product of a correspondingavirulence (Avr) gene in the pathogen. A plant line can be transformedwith cloned resistance gene to engineer plants that are resistant tospecific pathogen strains. See, for example Jones et al., Science266:789 (1994) (cloning of the tomato Cf-9 gene for resistance toCladosporium fulvum); Martin et al., Science 262:1432 (1993) (tomato Ptogene for resistance to Pseudomonas syringae pv. Tomato encodes a proteinkinase); Mindrinos et al., Cell 78:1089 (1994) (Arabidopsis RSP2 genefor resistance to Pseudomonas syringae).

[0156] B. A Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon. See, for example, Geiser et al.,Gene 48:109 (1986), who disclose the cloning and nucleotide sequence ofa Bt ä-endotoxin gene. Moreover, DNA molecules encoding ä-endotoxingenes can be purchased from American Type Culture Collection, Manassas,Va., for example, under ATCC Accession Nos. 40098, 67136, 31995 and31998.

[0157] C. A lectin. See, for example, the disclose by Van Damme et al.,Plant Molec. Biol. 24:25 (1994), who disclose the nucleotide sequencesof several Clivia miniata mannose-binding lectin genes.

[0158] D. A vitamin-binding protein such as avidin. See PCT applicationUS93/06487, the contents of which are hereby incorporated by reference.The application teaches the use of avidin and avidin homologues aslarvicides against insect pests.

[0159] E. An enzyme inhibitor, for example, a protease or proteinaseinhibitor or an amylase inhibitor. See, for example, Abe et al., J.Biol. Chem. 262:16793 (1987) (nucleotide sequence of rice cysteineproteinase inhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993)(nucleotide sequence of cDNA encoding tobacco proteinase inhibitor 1),Sumitani et al., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotidesequence of Streptomyces nitrosporeus á-amylase inhibitor).

[0160] F. An insect-specific hormone or pheromone such as an ecdysteroidand juvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

[0161] G. An insect-specific peptide or neuropeptide which, uponexpression, disrupts the physiology of the affected pest. For example,see the disclosures of Regan, J. Biol. Chem. 269:9 (1994) (expressioncloning yields DNA coding for insect diuretic hormone receptor), andPratt et al., Biochem. Biophys. Res. Comm. 163:1243 (1989) (anallostatin is identified in Diploptera puntata). See also U.S. Pat. No.5,266,317 to Tomalski et al., who disclose genes encodinginsect-specific, paralytic neurotoxins.

[0162] H. An insect-specific venom produced in nature by a snake, awasp, etc. For example, see Pang et al., Gene 116:165 (1992), fordisclosure of heterologous expression in plants of a gene coding for ascorpion insectotoxic peptide.

[0163] I. An enzyme responsible for a hyper accumulation of amonterpene, a sesquiterpene, a steroid, hydroxamic acid, aphenylpropanoid derivative or another non-protein molecule withinsecticidal activity.

[0164] J. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

[0165] K. A molecule that stimulates signal transduction. For example,see the disclosure by Botella et al., Plant Molec. Biol. 24:757 (1994),of nucleotide sequences for mung lettuce calmodulin cDNA clones, andGriess et al., Plant Physiol. 104:1467 (1994), who provide thenucleotide sequence of a maize calmodulin cDNA clone.

[0166] L. A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

[0167] M. A membrane permease, a channel former or a channel blocker.For example, see the disclosure of Jaynes et al., Plant Sci 89:43(1993), of heterologous expression of a cecropin-â, lytic peptide analogto render transgenic tobacco plants resistant to Pseudomonassolanacearum.

[0168] N. A viral-invasive protein or a complex toxin derived therefrom.For example, the accumulation of viral coat proteins in transformedplant cells imparts resistance to viral infection and/or diseasedevelopment effected by the virus from which the coat protein gene isderived, as well as by related viruses. See Beachy et al., Ann. rev.Phytopathol. 28:451 (1990). Coat protein-mediated resistance has beenconferred upon transformed plants against alfalfa mosaic virus, cucumbermosaic virus, tobacco streak virus, potato virus X, potato virus Y,tobacco etch virus, tobacco rattle virus and tobacco mosaic virus. Id.

[0169] O. An insect-specific antibody or an immunotoxin derivedtherefrom. Thus, an antibody targeted to a critical metabolic functionin the insect gut would inactivate an affected enzyme, killing theinsect. Cf. Taylor et al., Abstract #497, Seventh Int'l Symposium onMolecular Plant-Microbe Interactions (Edinburgh, Scotland) (1994)(enzymatic inactivation in transgenic tobacco via production ofsingle-chain antibody fragments).

[0170] P. A virus-specific antibody. See, for example, Tavladoraki etal., Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

[0171] Q. A developmental-arrestive protein produced in nature by apathogen or a parasite. Thus, fungal endo á-1, 4-D-polygalacturonasesfacilitate fungal colonization and plant nutrient release bysolubilizing plant cell wall homo-á-1,4-D-galacturonase. See Lamb etal., Bio/Technology 10:1436 (1992). The cloning and characterization ofa gene which encodes a lettuce endopolygalacturonase-inhibiting proteinis described by Toubart et al., Plant J. 2:367 (1992).

[0172] R. A development-arrestive protein produced in nature by a plant.For example, Logemann et al., Bioi/Technology 10:305 (1992), have shownthat transgenic plants expressing the barley ribosome-inactivating genehave an increased resistance to fungal disease.

[0173] R. A lettuce mosaic potyvirus (LMV) coat protein gene introducedinto Lactuca Sativa in order to increase its resistance to LMVinfection. See Dinant et al., Molecular Breeding. 1997, 3: 1, 75-86.

[0174] 2. Genes That Confer Resistance to a Herbicide, For Example:

[0175] A. A herbicide that inhibits the growing point or meristem, suchas an imidazalinone or a sulfonylurea. Exemplary genes in this categorycode for mutant ALS and AHAS enzyme as described, for example, by Lee etal., EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively.

[0176] B. Glyphosate (resistance impaired by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase, PAT and Streptomyces hygroscopicusphosphinothricin-acetyl transferase, bar, genes), and pyridinoxy orphenoxy propionic acids and cycloshexones (ACCase inhibitor-encodinggenes). See, for example, U.S. Pat. No. 4,940,835 to Shah, et al., whichdiscloses the nucleotide sequence of a form of EPSP which can conferglyphosate resistance. A DNA molecule encoding a mutant aroA gene can beobtained under ATCC accession number 39256, and the nucleotide sequenceof the mutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai. Seealso Umaballava-Mobapathie in Transgenic Research. 1999, 8: 1, 33-44that discloses lactuca sativa resistant to glufosinate. European patentapplication No. 0 333 033 to Kumada et al., and U.S. Pat. No. 4,975,374to Goodman et al., disclose nucleotide sequences of glutamine synthetasegenes which confer resistance to herbicides such as L-phosphinothricin.The nucleotide sequence of a phosphinothricin-acetyl-transferase gene isprovided in European application No. 0 242 246 to Leemans et al.,DeGreef et al., Bio/Technology 7:61 (1989), describe the production oftransgenic plants that express chimeric bar genes coding forphosphinothricin acetyl transferase activity. Exemplary of genesconferring resistance to phenoxy propionic acids and cycloshexones, suchas sethoxydim and haloxyfop are the Acc1-S1, Acc1-S2 and Acc1-S3 genesdescribed by Marshall et al., Theor. Appl. Genet. 83:435 (1992).

[0177] C. A herbicide that inhibits photosynthesis, such as a triazine(psbA and gs+ genes) and a benzonitrile (nitrilase gene). Przibilla etal., Plant Cell 3:169 (1991), describe the transformation ofChlamydomonas with plasmids encoding mutant psbA genes. Nucleotidesequences for nitrilase genes are disclosed in U.S. Pat. No. 4,810,648to Stalker, and DNA molecules containing these genes are available underATCC Accession Nos. 53435, 67441, and 67442. Cloning and expression ofDNA coding for a glutathione S-transferase is described by Hayes et al.,Biochem. J. 285:173 (1992).

[0178] 3. Genes that Confer or Contribute to a Value-Added Trait, suchas:

[0179] A. Increased iron content of the lettuce, for example bytransforming a plant with a soybean ferritin gene as decribed in Goto etal., Acta Horticulturae. 2000, 521, 101-109. Parallel to the improvediron content enhanced growth of transgenic lettuces was also observed inearly development stages.

[0180] B. Decreased nitrate content of leaves, for example bytransforming a lettuce with a gene coding for a nitrate reductase. Seefor example Curtis et al., Plant Cell Report. 1999, 18: 11, 889-896.

[0181] C. Increased sweetness of the lettuce by transferring a genecoding for monellin, that elicits a flavor 100000 times sweeter thansugar on a molar basis. See Penarrubia et al., Biotechnology. 1992, 10:5, 561-564.

[0182] Numerous methods for plant transformation have been developed,including biological and physical, plant transformation protocols. See,for example, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, GlickB. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993)pages 89-119.

[0183] A. Agrobacterium-Mediated Transformation

[0184] One method for introducing an expression vector into plants isbased on the natural transformation system of Agrobacterium. See, forexample, Horsch et al., Science 227:1229 (1985). Curtis et al., Journalof Experimental Botany. 1994, 45: 279, 1441-1449, Torres et al., Plantcell Tissue and Organic Culture. 1993, 34: 3, 279-285, Dinant et al.,Molecular Breeding. 1997, 3: 1, 75-86. A. tumefaciens and A. rhizogenesare plant pathogenic soil bacteria which genetically transform plantcells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes,respectively, carry genes responsible for genetic transformation of theplant. See, for example, Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991).Descriptions of Agrobacterium vector systems and methods forAgrobacterium-mediated gene transfer are provided by Gruber et al.,supra, Miki et al., supra, and Moloney et al., Plant Cell Reports 8:238(1989). See also, U.S. Pat. No. 5,591,616 issued Jan. 7, 1997.

[0185] B. Direct Gene Transfer

[0186] Despite the fact the host range for Agrobacterium-mediatedtransformation is broad, some major cereal or vegetable crop species andgymnosperms have generally been recalcitrant to this mode of genetransfer, even though some success has recently been achieved in riceand corn. Hiei et al., The Plant Journal 6:271-282 (1994) and U.S. Pat.No. 5,591,616 issued Jan. 7, 1997. Several methods of planttransformation, collectively referred to as direct gene transfer, havebeen developed as an alternative to Agrobacterium-mediatedtransformation.

[0187] A generally applicable method of plant transformation ismicroprojectile-mediated transformation wherein DNA is carried on thesurface of microprojectiles measuring 1 to 4 μm. The expression vectoris introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate plant cell walls and membranes. Russell, D. R.,et al. Pl. Cell. Rep. 12(January 3), 165-169 (1993), Aragao, F. J. L.,et al. Plant Mol. Biol. 20(October 2), 357-359 (1992), Aragao, F. J. L.,et al. Pl. Cell. Rep. 12(July 9), 483-490 (1993). Aragao Theor. Appl.Genet. 93:142-150 (1996), Kim, J.; Minamikawa, T. Plant Science 117:131-138 (1996), Sanford et al., Part. Sci. Technol. 5:27 (1987),Sanford, J. C., Trends Biotech. 6:299 (1988), Klein et al.,Bio/Technology 6:559-563 (1988), Sanford, J. C., Physiol Plant 7:206(1990), Klein et al., Biotechnology 10:268 (1992)

[0188] Another method for physical delivery of DNA to plants issonication of target cells. Zhang et al., Bio/Technology 9:996 (1991).Alternatively, liposome or spheroplast fusion have been used tointroduce expression vectors into plants. Deshayes et al., EMBO J.,4:2731 (1985), Christou et al., Proc Natl. Acad. Sci. U.S.A. 84:3962(1987). Direct uptake of DNA into protoplasts using CaCl₂ precipitation,polyvinyl alcohol or poly-L-omithine have also been reported. Hain etal., Mol. Gen. Genet. 199:161 (1985) and Draper et al., Plant CellPhysiol. 23:451 (1982). Electroporation of protoplasts and whole cellsand tissues have also been described. Saker, M.; Kuhne, T. BiologiaPlantarum 40(4): 507-514 (1997/98), Donn et al., In Abstracts of VIIthInternational Congress on Plant Cell and Tissue Culture IAPTC, A2-38, p53 (1990); D'Halluin et al., Plant Cell 4:1495-1505 (1992) and Spenceret al., Plant Mol. Biol. 24:51-61 (1994). See also Chupean et al.,Biotechnology. 1989, 7: 5, 503-508.

[0189] Following transformation of lettuce target tissues, expression ofthe above-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

[0190] The foregoing methods for transformation would typically be usedfor producing a transgenic line. The transgenic line could then becrossed, with another (non-transformed or transformed) line, in order toproduce a new transgenic lettuce line. Alternatively, a genetic traitwhich has been engineered into a particular lettuce cultivar using theforegoing transformation techniques could be moved into another lineusing traditional backcrossing techniques that are well known in theplant breeding arts. For example, a backcrossing approach could be usedto move an engineered trait from a public, non-elite inbred line into anelite inbred line, or from an inbred line containing a foreign gene inits genome into an inbred line or lines which do not contain that gene.As used herein, “crossing” can refer to a simple X by Y cross, or theprocess of backcrossing, depending on the context.

[0191] When the term lettuce plant, cultivar or lettuce line are used inthe context of the present invention, this also includes any single geneconversions of that line. The term single gene converted plant as usedherein refers to those lettuce plants which are developed by a plantbreeding technique called backcrossing wherein essentially all of thedesired morphological and physiological characteristics of a cultivarare recovered in addition to the single gene transferred into the linevia the backcrossing technique. Backcrossing methods can be used withthe present invention to improve or introduce a characteristic into theline. The term backcrossing as used herein refers to the repeatedcrossing of a hybrid progeny back to one of the parental lettuce plantsfor that line. The parental lettuce plant which contributes the gene forthe desired characteristic is termed the nonrecurrent or donor parent.This terminology refers to the fact that the nonrecurrent parent is usedone time in the backcross protocol and therefore does not recur. Theparental cantaloupe plant to which the gene or genes from thenonrecurrent parent are transferred is known as the recurrent parent asit is used for several rounds in the backcrossing protocol (Poehiman &Sleper, 1994; Fehr, 1987). In a typical backcross protocol, the originalcultivar of interest (recurrent parent) is crossed to a second line(nonrecurrent parent) that carries the single gene of interest to betransferred. The resulting progeny from this cross are then crossedagain to the recurrent parent and the process is repeated until alettuce plant is obtained wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the singletransferred gene from the nonrecurrent parent.

[0192] The selection of a suitable recurrent parent is an important stepfor a successful backcrossing procedure. The goal of a backcrossprotocol is to alter or substitute a single trait or characteristic inthe original line. To accomplish this, a single gene of the recurrentcultivar is modified or substituted with the desired gene from thenonrecurrent parent, while retaining essentially all of the rest of thedesired genetic, and therefore the desired physiological andmorphological, constitution of the original line. The choice of theparticular nonrecurrent parent will depend on the purpose of thebackcross, one of the major purposes is to add some commerciallydesirable, agronomically important trait to the plant. The exactbackcrossing protocol will depend on the characteristic or trait beingaltered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired characteristic has beensuccessfully transferred.

[0193] Many single gene traits have been identified that are notregularly selected for in the development of a new line but that can beimproved by backcrossing techniques. Single gene traits may or may notbe transgenic, examples of these traits include but are not limited to,herbicide resistance, resistance for bacterial, fungal, or viraldisease, insect resistance, enhanced nutritional quality, industrialusage, yield stability and yield enhancement. These genes are generallyinherited through the nucleus. Several of these single gene traits aredescribed in U.S. Pat. Nos. 5,777,196; 5,948,957 and 5,969,212, thedisclosures of which are specifically hereby incorporated by reference.

TABLES

[0194] In the tables that follow, the traits and characteristics oflettuce cultivar HMX 7555X are given compared to other check cultivars.

[0195] The first column lists the variety tested.

[0196] The second column shows head weight in grams.

[0197] The head length in centimeters is shown in column 3.

[0198] The core diameter in centimeters is shown in column 4.

[0199] The core length in centimeters is shown in column 5. TABLE 1Overall Comparisons Lettuce named HMX 7555 vs Checks Location: 1999 inYUMA, Arizona Head Head Variety weight length Core diameter Core lengthHMX 7555 1221.1 35 3.46 8.1 Conquistador 1177.8 34.55 3.51 10.07 GreenForest 1042.2 34.1 3.64 8.41 Green Towers 1047.8 34.9 3.45 7.57

[0200] TABLE 2 Overall Comparisons Lettuce named HMX 7555 vs ChecksLocation: 2000 in San Juan Bautista, California Head Head Variety weightlength Core diameter Core length HMX 7555 1837 33.5 4.56 11.94 GreenTowers 1689.7 32.65 4.43 10.06 Darkland Cos 1523.4 33.7 4.29 9.97

DEPOSIT INFORMATION

[0201] A deposit of the lettuce cultivar seed of this invention ismaintained by Harris Moran Seed Company, 100 Breen Road, San JuanBautista, Calif. 95045, USA. Access to this deposit will be availableduring the pendency of this application to persons determined by theCommissioner of Patent and Trademarks to be entitled thereto under 37CRF 1.14 and 35 USC 122. Upon allowance of any claims in thisapplication, all restrictions on the availability to the public of thevariety will be irrevocably removed by affording access to a deposit ofat least 2,500 seeds of the same variety with the American Type CultureCollection (ATCC), 10801 University Boulevard, Manassas, Va. 20110.

[0202] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity andunderstanding. However, it will be obvious that certain changes andmodifications such as single gene modifications and mutations,somaclonal variants, variant individuals selected from large populationsof the plants of the instant line and the like may be practiced withinthe scope of the invention, as limited only by the scope of the appendedclaims.

What is claimed is:
 1. A Lactuca sativa romaine lettuce seed designatedHMX 7555, wherein a sample of said seed has been deposited under ATCCAccession No. _____.
 2. A plant, or its parts, produced by growing theseed of claim
 1. 3. Pollen of the plant of claim
 2. 4. An ovule of theplant of claim
 2. 5. A Lactuca sativa romaine lettuce plant having allof the physiological and morphological characteristics of the lettuceplant of claim 2, or its parts.
 6. A tissue culture of regenerable cellsof a lettuce plant of variety HMX 7555, wherein the tissue regeneratesplants capable of expressing all the morphological and physiologicalcharacteristics of Lactuca sativa romaine lettuce line HMX 7555,representative seeds having been deposited under ATCC number ______. 7.The tissue culture of claim 6, selected from the group consisting ofprotoplast and calli, wherein the regenerable cells are derived fromembryo, meristematic cells, leaves, pollen, embryo, root, root tips,stems, anther, flowers or seeds.
 8. A Lactuca sativa romaine lettuceplant regenerated from the tissue culture of claim 6, capable ofexpressing all the morphological and physiological characteristics ofLactuca sativa romaine lettuce plant HMX 7555, representative seedshaving been deposited under ATCC number ______.
 9. A method forproducing a lettuce seed comprising crossing a first parent lettuceplant with a second parent lettuce plant and harvesting the resultanthybrid lettuce seed, wherein said first or second parent lettuce plantis the Lactuca sativa romaine lettuce plant of claim
 2. 10. A hybridlettuce seed produced by the method of claim
 9. 11. A hybrid lettuceplant, or its parts, produced by growing said hybrid lettuce seed ofclaim
 10. 12. A lettuce seed produced by growing said hybrid lettuceplant of claim 11 and harvesting the resultant lettuce seed.
 13. Amethod for producing a hybrid lettuce seed comprising crossing anLactuca sativa romaine lettuce plant according to claim 2 with another,different lettuce plant.
 14. A hybrid lettuce seed produced by themethod of claim
 13. 15. A hybrid lettuce plant, or its parts, producedby growing said hybrid lettuce seed of claim
 14. 16. A lettuce seedproduced by growing said hybrid lettuce plant of claim 15 and harvestingthe resultant seed.
 17. A method for producing a HMX 7555-derivedlettuce plant, comprising: a) crossing lettuce line HMX 7555, a sampleof seed of said line having been deposited under ATCC accession number______, with a second lettuce plant to yield progeny lettuce seed; andb) growing said progeny lettuce seed, under plant growth conditions, toyield said HMX 7555-derived lettuce plant.
 18. A HMX 7555-derivedlettuce plant, or parts thereof, produced by the method of claim 17,said HMX 7555-derived lettuce plant expressing a combination of at leasttwo HMX 7555 traits selected from the group consisting of: dark andglossy leaves, a summer maturity of 98-108 days, superior weight,resistance to downy mildew pathotype 1, intermediate resistance toSclerotinia rot and adapted to the Southwest, West coast and Northeastgrowing regions of the USA.
 19. The method of claim 17, furthercomprising: c) crossing said HMX 7555-derived lettuce plant with itselfor another lettuce plant to yield additional HMX 7555-derived progenylettuce seed; d) growing said progeny lettuce seed of step (c) underplant growth conditions, to yield HMX 7555-derived lettuce plant; e)repeating the crossing and growing steps of (c) and (d) from 0 to 7times to generate further HMX 7555-derived lettuce plant.
 20. A furtherHMX 7555-derived lettuce plant or parts thereof, produced by the methodof claim 19, said HMX 7555-derived lettuce plant expressing acombination of at least two HMX 7555traits selected from the groupconsisting of: dark and glossy leaves, a summer maturity of 98-108 days,superior weight, resistance to downy mildew pathotype I, intermediateresistance to Sclerotinia rot and adapted to the Southwest, West coastand Northeast growing regions of the USA.
 21. The method of claim 17,still further comprising utilizing plant tissue culture methods toderive progeny of said HMX 7555-derived lettuce plant.
 22. A further HMX7555-derived lettuce plant or parts thereof, produced by the method ofclaim 21, said HMX 7555-derived lettuce plant expressing a combinationof at least two HMX 7555 traits selected from the group consisting of:dark and glossy leaves, a summer maturity of 98-108 days, superiorweight, resistance to downy mildew pathotype I, intermediate resistanceto Sclerotinia rot and adapted to the Southwest, West coast andNortheast growing regions of the USA.
 23. The Lactuca sativa romainelettuce plant, or parts thereof, of claim 2, wherein the plant or partsthereof have been transformed so that its genetic material contains oneor more transgenes operably linked to one or more regulatory elements.24. A method for producing a lettuce plant that contains in its geneticmaterial one or more transgenes, comprising crossing the Lactuca sativalettuce plant of claim 23 with either a second plant of another lettuceline, or a non-transformed lettuce plant of the line HMX 7555, so thatthe genetic material of the progeny that result from the cross containsthe transgene(s) operably linked to a regulatory element.
 25. Lettuceplants, or parts thereof, produced by the method of claim
 24. 26. Amethod for developing a lettuce plant in a lettuce plant breedingprogram using plant breeding techniques which include employing alettuce plant, or its parts, as a source of plant breeding materialcomprising: using the Lactuca sativa lettuce plant, or its parts, ofclaim 2 as a source of said breeding material.
 27. The lettuce plantbreeding program of claim 26 wherein plant breeding techniques areselected from the group consisting of: recurrent selection,backcrossing, pedigree breeding, restriction fragment lengthpolymorphism enhanced selection, genetic marker enhanced selection, andtransformation.
 28. A lettuce plant, or parts thereof, produced by themethod of claim 26, said lettuce plant expressing a combination of atleast two HMX 7555 traits selected from the group: dark and glossyleaves, a summer maturity of 98-108 days, superior weight, resistance todowny mildew pathotype 1, intermediate resistance to Sclerotinia rot andadapted to the Southwest, West coast and Northeast growing regions ofthe USA.
 29. The Lactuca sativa lettuce plant of claim 5, furthercomprising a single gene conversion.
 30. The single gene conversion ofthe Lactuca sativa lettuce plant of claim 29, where the gene is selectedfrom the group consisting of: a transgenic gene, a dominant allele, anda recessive allele.
 31. The single gene conversion of the Lactuca sativalettuce plant of claim 29, where the gene confers a characteristicselected from the group consisting of: herbicide resistance, insectresistance, resistance to bacterial, fungal, or viral disease,endosperm, and improved nutritional quality.
 32. A lettuce plant, orpart thereof, wherein at least one ancestor of said lettuce plant is theLactuca sativa lettuce plant of claim 2, said lettuce plant expressing acombination of at least two HMX 7555 traits selected from the groupconsisting of: dark and glossy leaves, a summer maturity of 98-108 days,superior weight, resistance to downy mildew pathotype I, intermediateresistance to Sclerotinia rot and adapted to the Southwest, West coastand Northeast growing regions of the USA.